This invention is generally in the field of peptide-based vaccines to control atherogenic activity in the circulatory system of humans and other animals. In particular, this invention provides compositions and methods for providing endogenous means to inhibit the activity of endogenous cholesteryl ester transfer protein (CETP) and to effectively modulate the relative levels of lipoproteins to produce a condition correlated with a reduced risk of cardiovascular disease, such as atherosclerosis.
Cholesterol circulates through the body predominantly as components of lipoprotein particles (lipoproteins), which are composed of a protein portion, called apolipoproteins (Apo) and various lipids, including phospholipids, triglycerides, cholesterol and cholesteryl esters. There are ten major classes of apolipoproteins: Apo A-I, Apo A-II, Apo-IV, Apo B-48, Apo B-100, Apo C-I, Apo C-II, Apo C-III, Apo D, and Apo E. Lipoproteins are classified by density and composition. For example, high density lipoproteins (HDL), one function of which is to mediate transport of cholesterol from peripheral tissues to the liver, have a density usually in the range of approximately 1.063-1.21 g/ml. HDL contain various amounts of Apo A-I, Apo A-II, Apo C-I, Apo C-II, Apo C-III, Apo D, Apo E, as well as various amounts of lipids, such as cholesterol, cholesteryl esters, phospholipids, and triglycerides.
In contrast to HDL, low density lipoproteins (LDL), which generally have a density of approximately 1.019-1.063 g/ml, contain Apo B-100 in association with various lipids. In particular, the amounts of the lipids cholesterol and cholesteryl esters are considerably higher in LDL than in HDL, when measured as a percentage of dry mass. LDL are particularly important in delivering cholesterol to peripheral tissues.
Very low density lipoproteins (VLDL) have a density of approximately 0.95-1.006 g/ml and also differ in composition from other classes of lipoproteins both in their protein and lipid content. VLDL generally have a much higher amount of triglycerides (TG) than do HDL or LDL and are particularly important in delivering endogenously synthesized triglycerides from liver to adipose and other tissues. The features and functions of various lipoproteins have been reviewed (see, for example, Mathews, C. K. and van Holde, K. E., Biochemistry, pp. 574-576, 626-630 (The Benjamin/Cummings Publishing Co., Redwood City, Calif., 1990); Havel, R. J., et al., et al., xe2x80x9cIntroduction: Structure and metabolism of plasma lipoproteinsxe2x80x9d, In The Metabolic Basis of Inherited Disease, 6th ed., pp. 1129-1138 (Scriver, C. R., et al., eds.) (McGraw-Hill, Inc., New York, 1989); Zannis, V. I., et al., xe2x80x9cGenetic mutations affecting human lipoproteins, their receptors, and their enzymesxe2x80x9d, In Advances in Human Genetics Vol. 21, pp. 145-319 (Plenum Press, New York, 1993)).
Decreased susceptibility to cardiovascular disease, such as atherosclerosis, is generally correlated with increased absolute levels of circulating HDL and also increased levels of HDL relative to circulating levels of lower density lipoproteins such as VLDL and LDL (see, e.g., Gordon, D. J., et al., N. Engl. J. Med., 321: 1311-1316 (1989); Castelli, W. P., et al., J. Am. Med. Assoc., 256: 2835-2838 (1986); Miller, N. E., et al., Am. Heart J., 113: 589-597 (1987); Tall, A. R, J. Clin. Invest., 89: 379-384 (1990); Tall, A. R., J. Internal Med, 237: 5-12 (1995)).
Cholesteryl ester transport protein (CETP) mediates the transfer of cholesteryl esters from HDL to TG-rich lipoproteins such as VLDL and LDL, and also the reciprocal exchange of TG from VLDL to HDL (Tall, A. R., J. Internal Med., 237: 5-12 (1995); Tall, A. R., J. Lipid Res., 34: 1255-1274 (1993); Hesler, C. B., et al., J. Biol. Chem., 262: 2275-2282 (1987); Quig, D. W. et al., Ann. Rev. Nutr., 10: 169-193 (1990)). CETP may play a role in modulating the levels of cholesteryl esters and TG associated with various classes of lipoproteins. A high CETP cholesteryl ester transfer activity has been correlated with increased levels of LDL-associated cholesterol and VLDL-associated cholesterol, which in turn are correlated with increased risk of cardiovascular disease (see, e.g., Tato, F., et al., Arterioscler. Thromb. Vascular Biol., 15: 112-120 (1995)).
Hereinafter, LDL-C will be used to refer to total cholesterol, including cholesteryl esters and/or unesterified cholesterol, associated with low density lipoprotein. VLDL-C will be used to refer to total cholesterol, including cholesteryl esters and/or unesterified cholesterol associated with very low density lipoprotein. HDL-C will be used to refer to total cholesterol, including cholesteryl esters and/or unesterified cholesterol, associated with high density lipoprotein.
CETP isolated from human plasma is a hydrophobic glycoprotein having 476 amino acids and a molecular weight of approximately 66,000 to 74,000 daltons on sodium dodecyl sulfate (SDS)-polyacrylamide gels (Albers, J. J., et al., Arteriosclerosis, 4: 49-58 (1984); Hesler, C. B., et al., J. Biol. Chem., 262: 2275-2282 (1987); Jarnagin, S. S., et al., Proc. Natl. Acad. Sci. USA, 84: 1854-1857 (1987)). A cDNA encoding human CETP has been cloned and sequenced (Drayna, D., et al., Nature, 327: 632-634 (1987)). CETP has been shown to bind CE, TG, phospholipids (Barter, P. J. et al., J. Lipid Res., 21:238-249 (1980)), and lipoproteins (see, e.g., Swenson, T. L., et al., J. Biol. Chem., 264: 14318-14326 (1989)). More recently, the region of CETP defined by the carboxyl terminal 26 amino acids, and in particular amino acids 470 to 475, has been shown to be especially important for neutral lipid binding involved in neutral lipid transfer (Hesler, C. B., et al., J. Biol. Chem., 263: 5020-5023 (1988)), but not phospholipid binding (see, Wang, S., et al., J. Biol. Chem., 267: 17487-17490 (1992); Wang, S., et al., J. Biol. Chem., 270: 612-618 (1995)).
A monoclonal antibody (Mab), TP2 (formerly designated 5C7 in the literature), has been produced which inhibits completely the cholesteryl ester and TG transfer activity of CETP, and to a lesser extent the phospholipid transfer activity (Hesler, C. B., et al., J. Biol. Chem., 263: 5020-5023 (1988)). The epitope of TP2 was localized to the carboxyl terminal 26 amino acids, i.e., the amino acids from arginine-451 to serine-476, of the 74,000 dalton human CETP molecule (see, Hesler, C. B., et al., (1988)). TP2 was reported to inhibit CETP activity in human plasma in vitro (Yen, F. T., et al., J. Clin. Invest., 83: 2018-2024 (1989)). Further analysis of the region of CETP bound by TP2 revealed that amino acids between phenylalanine-463 and leucine-475 are necessary for TP2 binding and for neutral lipid (e.g., cholesteryl ester) transfer activity (see, Wang, S., et al., 1992).
A number of in vivo studies utilizing animal models or humans have indicated that CETP activity can affect the level of circulating cholesterol-containing HDL. Increased CETP cholesteryl ester transfer activity can produce a decrease in HDL-C levels relative to LDL-C and/or VLDL-C (where HDL-C, LDL-C, and VLDL-C refer generally to cholesteryl ester and/or unesterified cholesterol associated with HDL, LDL, and VLDL, respectively) levels which in turn is correlated with an increased susceptibility to atherosclerosis. Injection of partially purified human CETP into rats (which normally lack CETP activity), resulted in a shift of cholesteryl ester from HDL to VLDL, consistent with CETP-promoted transfer of cholesteryl ester from HDL to VLDL (Ha, Y. C., et al., Biochim. Biophys. Acta, 833: 203-211 (1985); Ha, Y. C., et al., Comp. Biochem. Physiol., 83B: 463-466 (1986); Gavish, D., et al., J. Lipid Res., 28: 257-267 (1987)). Transgenic mice expressing human CETP were reported to exhibit a significant decrease in the level of cholesterol associated with HDL (see, e.g., Hayek, T., et al., J. Clin. Invest., 90: 505-510 (1992); Breslow, J. L., et al., Proc. Natl. Acad. Sci. USA, 90: 8314-8318 (1993)). Furthermore, whereas wild-type mice are normally highly resistant to atherosclerosis (Breslow, J. L., et al., Proc. Natl. Acad. Sci. USA, 90: 8314-8318 (1993)), transgenic mice expressing a simian CETP were reported to have an altered distribution of cholesterol associated with lipoproteins, namely, elevated levels of LDL-C and VLDL-C and decreased levels of HDL-C (Marotti K. R., et al., Nature, 364: 73-75 (1993)). Transgenic mice expressing simian CETP also were susceptible to dietary-induced severe athersclerosis compared to non-expressing control mice (Marotti et al., id.). Intravenous infusion of anti-human CETP monoclonal antibodies (Mab) into hamsters and rabbits inhibited CETP activity in vivo and resulted in significantly increased levels of HDL-C levels, decreased levels of HDL-TG, and increased HDL size; again implicating a critical role for CETP in the distribution of cholesterol in circulating lipoproteins (Gaynor, B. J., et al., Atherosclerosis, 110: 101-109 (1994) (hamsters); Whitlock, M. E., et al., J. Clin. Invest., 84: 129-137 (1989) (rabbits)).
CETP deficiency has also been studied in humans. For example, in certain familial studies in Japan, siblings that were homozygous for non-functional alleles of the CETP gene had no detectable CETP activity. Virtually no atherosclerotic plaques were exhibited by these individuals, who also showed a trend toward longevity in their families (see, e.g., Brown, M. L., et al., Nature, 342: 448-451 (1989); Inazu, A., et al., N. Engl. J. Med., 323: 1234-1238 (1990); Bisgaier, C. L., et al., J. Lipid Res., 32: 21-23 (1991)). Such homozygous CETP-deficient individuals also were shown to have an anti-atherogenic lipoprotein profile as evidenced by elevated levels of circulating HDL rich in cholesteryl ester, as well as overall elevated levels of HDL, and exceptionally large HDL, i.e., up to four to six times the size of normal HDL (Brown, M. L., et al., 1989, p. 451). The frequency of this mutation in Japan is relatively high, and may account for an elevated level of HDL in a significant fraction of the Japanese population.
The above studies indicate that CETP plays a major role in transferring cholesteryl ester from HDL to VLDL and LDL, and thereby in altering the relative profile of circulating lipoproteins to one which is associated with an increased risk of cardiovascular disease (e.g., decreased levels of HDL-C and increased levels of VLDL-C and LDL-C). Taken together, the current evidence suggests that increased levels of CETP activity may be predictive of increased risk of cardiovascular disease. Modulation or inhibition of endogenous CETP activity is thus an attractive therapeutic method for modulating the relative levels of lipoproteins to reduce or prevent the progression of or to induce regression of cardiovascular diseases, such as atherosclerosis.
It would be advantageous, therefore, to discover compounds and methods to control CETP activity which would be helpful in preventing or treating cardiovascular disease. To be an effective pharmacological therapeutic, a compound when administered to a significant majority of recipients, ideally, would not elicit an immune response which neutralizes the beneficial activity or effect of the therapeutic compound, must not promote a hypersensitive state in the individual receiving the therapeutic compound, and must not produce untoward side effects. It would also be advantageous if such compounds and methods avoided the necessity for continuous or frequently repeated treatments.
This invention provides compounds and methods useful for the modulation or inhibition of cholesteryl ester transfer protein (CETP) activity. In particular, vaccine peptides are described which, when administered to a mammal, raise an antibody response against the mammal""s own endogenous CETP. Such vaccine peptides comprise a helper T cell epitope portion comprising a universal immunogenic helper T cell epitope, linked, preferably covalently, to a B cell epitope portion comprising a carboxyl terminal portion of human CETP protein that is involved in a neutral lipid binding or a transfer activity of CETP. In a preferred embodiment, the helper T cell epitope portion of a vaccine peptide of this invention is derived from an amino acid sequence of a universally immunogenic helper T cell epitope, such as those found in tetanus and diptheria toxoids, or in antigenic peptides known from pertussis vaccine, Bacile Calmette-Guerin (BCG), polio vaccine, measles vaccine, mumps vaccine, rubella vaccine, purified protein derivative (PPD) of tuberculin, and keyhole limpet hemocyanin. Furthermore, various universal antigenic helper T cell epitopes may be linked to one another to form multiple universal antigenic helper T cell epitope portions of the vaccine peptides of this invention. In a more preferred embodiment of a vaccine peptide of this invention, an amino terminal cysteine residue is covalently linked to an amino acid sequence of a universal antigenic helper T cell epitope of tetanus toxoid forming the sequence C Q Y I K A N S K F I G I T E (amino acids 1 to 15 of SEQ ID NO:2), which is covalently linked to a B cell epitope portion of a vaccine peptide having the carboxyl terminal CETP amino acid sequence F G F P E H L L V D F L Q S L S (amino acids 16 to 31 of SEQ ID NO:2).
The peptides of this invention may also be linked to a common molecule to form peptide assemblies in which multiple copies of the peptides are arranged close to one another. Such multicopy (or multivalent) peptide assemblies may be more immunogenic, that is, produce a more effective immune response to endogenous CETP than vaccines comprising unassociated individual peptides. The vaccine compounds of this invention also may be used in combination with a pharmaceutically acceptable adjuvant.
The immunogenic vaccine peptides of this invention elicit the production of antibodies that are reactive with or recognize CETP. Administration of vaccine peptides to test animals resulted in a decline in the relative levels of total cholesterol and HDL-C. The elicited endogenous anti-CETP antibodies may promote a physiological condition correlated with decreased risk of cardiovascular disease, such as atherosclerosis.